A competitive immunoassay for the detection of esterolytic activity of antibodies and enzymes.
Screening of a large number of clones produced in a fusion is often the bottleneck in the isolation of catalytic antibodies. The usual approach requires two steps: clones are first selected for their high affinity to the antigen, and then the good binders are tested for their catalytic activity. To simplify this selection process, a competitive enzyme-linked immunosorbent assay (ELISA) has been developed that allows direct screening of the antibodies on the basis of their catalytic activity. In this assay, the product of the catalyzed reaction binds to an immobilized anti-product antibody in competition with a peroxidase-product conjugate. The screening assay has been developed for the antibody-catalyzed hydrolysis of esters of p-aminophenylacetic acid and has been tested on the porcine liver esterase (PLE)-catalyzed hydrolysis of the same substrates. This test allows the detection of product formation at the nanomolar level, while, in a typical assay, the catalytic activity of PLE can be traced down to 200 fmol of enzyme. Under standard conditions for the screening of hybridomas obtained from a fusion, the competitive ELISA allows detection of catalytic species with values of kcat > or = 5 x 10(-7) mol l-1 s-1 and kcat/kuncat > or = 50. While the assay has been designed for the selection of catalytic antibodies, other potential applications of this methodology are in the screening of libraries of engineered and designed enzymes and, in general, in the quantitative measurement of enzyme activity.
Benedetti
F
F
Dipartimento di Scienze Chimiche, Università di Trieste, Italy. benedett@dschsun1.univ.trieste.it
Berti
F
F
Flego
M
M
Resmini
M
M
Bastiani
E
E
D000818:Animals; D017151:Antibodies, Catalytic / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001667:Binding, Competitive; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000706:statistics & numerical data; D004798:Enzymes / Q000378:metabolism*; D004950:Esterases / Q000378:metabolism; D005069:Evaluation Studies as Topic; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D011817:Rabbits; D012680:Sensitivity and Specificity; D013552:Swine
D017151:Antibodies, Catalytic; D000911:Antibodies, Monoclonal; D004798:Enzymes; D004950:Esterases